Separation processes in which two immiscible or partially soluble liquid phases are brought into contact for the transfer of one or more components are referred to as liquid-liquid extraction or solvent extraction. In the simplest case, when two immiscible solvents containing solute are shaken in a separatory funnel and then separated, the solutes are partitioned between the two phases. The ratio of solute concentration in the upper phase to the lower is called the partition coefficient. If the partition coefficient of the two substances differ greatly, the only process needed for separation is a one-step operation. As the nature of the substances become more similar, the difference in their partition coefficients decrease, hence requiring multi-step extraction or separation. When done in countercurrent fashion, this technique is called "the countercurrent distribution method."
Another method of extraction is known as partition chromatography. This method involves a continuous partition process between moving and stationary phases. A variety of methods have been developed for partition chromatography which employ solid supports such as cellulose, silica, alumina, or glass in order to hold one phase stationary. The granular and porous nature of the solid support provides an enormous surface area in relation to the liquid volume and divides the free space into thousands of "plates." In each plate, the partition process is theoretically completed, thus yielding an efficiency as high as thousands of theoretical plates. However, the affinity of the solid supports for the solute can add an undesirable adsorption effect evidenced by tailing of the elution curves of the solutes. When one deals with a minute amount of biological components, adsorption can result in a significant loss or denaturation of samples in addition to contamination by foreign materials eluted from the support.
Another liquid-liquid extraction technique has also been developed which is known as countercurrent chromatography. This system is similar to the countercurrent distribution method in that the two immiscible phases pass through each other in a tubular space. However, it involves a continuous non-equilibrium partition process comparable to chromatography. It was developed to achieve a high efficiency chromatographic separation on both a preparative and analytical scale in the absence of solid supports. However, elimination of the solid support creates a number of problems as listed below:
1. How to keep the stationary phase in the column as the moving phase is steadily eluted. PA1 2. How to divide the column space into numerous partition units and reduce laminar flow spreading of the sample bands. PA1 3. How to increase interfacial area. PA1 4. How to mix each phase to reduce mass transfer resistance.
Several arrangements of countercurrent chromatography have been developed. In each system, a tubular column is made to form multiple traps to hold the stationary phase in a segmented pattern while a gravitational or centrifugal force maintains the two-phase states. Relative interface area is increased by decreasing the tubular diameter and/or increasing the number of the phase segments per unit length of the column. In some cases, effective mixing is accomplished by rotational or gyrational motion of the column, while the interface is held stable by gravity or centrifugal force.
Several methods of countercurrent chromatography have been disclosed in the article by Ito and Bowman in "Journal of Chromatographic Science," vol. 8, pages 315-323, June 1970. These methods include helix countercurrent chromatography, droplet countercurrent chromatography and rotation and gyration locular countercurrent chromatography.
In helix countecurrent chromatography, a horizontal helical tube is filled with one phase of a two-phase liquid system. The other phase is introduced at one end of the helix and passes through the first phase according to the vertical direction of flow resulting in alternate segments of the two phases. Continued flow causes displacement of the second phase only with respect to the stationary first phase. A liquid-liquid partition chromatographic system is thus established. Solutes introduced to either phase will undergo separation according to their relative partition coefficients in a manner analogous to that of conventional liquid chromatography but in the absence of a solid support. The force of gravity holds the lower phase stationary while the upper phase is forced therethrough. To enable the countercurrent process to take place inside a very small diameter tube having a maximum of turns, the enhancement of the gravitational field is necessitated. This is achieved by the use of a centrifuge.
In an article by Ito et al. in Analytical Chemistry, vol. 41, pages 1579-1584, October 1969, such a helix countercurrent chromatography method is described using a coil planet centrifuge. This apparatus induces a planetary motion to a helical tube in a manner such that the rotation of the helical tube is extremely slow in comparison with the revolution. In such a system, however, flow becomes difficult because of the need for the rotating seals; therefore, the separation is usually performed within a closed helical tube. Thus, problems of sample introduction and fractionation limit the practical use of the method.
More recently, two improved countercurrent chromatographic shemes have veen devised, both utilizing a coiled tube in a centrifugal field. One is the flow-through coil planet centrifuge with a vertical rotor, disclosed in U.S. Pat. No. 3,755,309, and the other, the elution centrifuge with a horizontal rotor, disclosed in U.S. Pat. No. 3,856,669. Experiments with various two-phase solvent systems have disclosed specific features inherent to each of these schemes. The advantage of the vertical rotor is that a high efficiency partitioning is achieved with a short separation time by providing a vigorous phase mixing. However, it fails to retain the stationary phase of some low interfacial tension phase systems such as polymer phase systems, due to intensive emulsification. On the other hand, the horizontal rotor provides a stable centrifugal force field to retain polymer phase systems but a longer separation time is generally required due to increased mass transfer resistance.